Multiplex labeling of cells for analysis of mixed cell populations by flow cytometry has progressed in various ways. Only a finite number of fluorescence emission colors of known organic fluorophores can be squeezed into the visible-near-UV-near-IR spectral regions in which flow cytometry measurements are made. The limitations have been dictated by the bandwidths of emission bands, the spectral overlap between these emission bands, and the excitation wavelength requirements. Up to eight colors requiring three laser lines have been introduced in various stages: two- to four-color [H. M. Shapiro in PRACTICAL FLOW CYTOMETRY, 3rd ed., Wiley-Liss, Inc., New York, N.Y., 1995, Chap. 7, p. 291]; five color [A. J. Beavis and K. J. Pennline, Cytometry, 15: 371-376 (1994); M. C. O'Brien et al, Cytometry, 21:76-83 (1995); M. Roederer et al, Cytometry, 21: 187-196 (1995)]; six color [M. Roederer et al, Cytometry, 24: 191-197 (1996)]; seven color for imaging [T. Ried et al, Proc. Natl. Acad. Sci. USA, 89: 1388-1392 (1992); A. Gothot et al, Cytometry, 24: 214-225 (1996)]; and eight color [M. Roederer et al, Tissue Antigens, 48: 485 (1996), abstract TC-6-02]. The six and seven color cases appear to represent the present upper limit for flow cytometry applications in which known organic dyes are used as fluorescent labels, since the eight-color example cannot as yet be considered to be of clinical significance due to severe overlap between emission bands of the fluorochromes.
As the upper limit in the number of usable colors was reached, other methods, based on fluorescence intensity differences, either intrinsic to analyzed cell populations or contrived by various means, have been described. Mutually exclusive pairs of targeted white blood cell populations with widely different, intrinsic numbers of receptors per cell can be labeled by a single color marker and analyzed by flow cytometry [U.S. Pat. No. 5,538,855, issued Jul. 23, 1996]. U.S. Pat. No. 4,499,052, issued Feb. 12, 1985 describes a method of distinguishing multiple subpopulations of cells by labeling specific antibodies with fluorescent polymers containing different, pre-selected ratios of fluorescein and rhodamine [see also, A. M. Saunders and C.-H. Chang, Ann. N. Y. Acad. Sci., 468:128 (1986)]. H. M. Shapiro in PRACTICAL FLOW CYTOMETRY, 1st ed., Alan R. Liss, Inc., pp. 127-128 (1985) describes a method using three different antibodies labeled with fluorochrome A, B, and a combination of A and B. U.S. Pat. No. 5,206,143, issued Apr. 27, 1993 describes saturated and sub-saturated amounts of marker mixed with the sample of cells. Quantitative differences in fluorescence intensity of one or two fluorochromes used for labeling cells were obtained. Each subset to be analyzed was labeled with a different amount of fluorochrome, exhibiting fluorescence intensities within a distinguishable range. Mixtures of fluorescein- and phycoerythrin-labeled and unlabeled antibodies were used to produce fluorescence intensity differences of several orders of magnitude among various cell subsets [P. K. Horan et al, Proc. Natl. Acad. Sci. USA, 83: 8361-8365 (1986)]. Use of this method to identify helper and suppressor/cytotoxic T cells, NK and B cells, and monocytes in whole blood was shown [C.-M. Liu et al, Am. J. Clin. Path, 92: 721-728 (1989)]. Further, eight leukocyte subsets in whole blood were analyzed with six monoclonal antibodies linked with one of three fluorochromes [P. Carayon et al, J. Immunol. Methods, 138: 257-264 (1991)].
Enhanced phycoerythrin (PE, excitation, 486-575 nm, emission, 568-590 nm) fluorescence intensities per marker for direct antibody-PE conjugates were obtained by substituting the direct conjugates with aminodextran-crosslinked conjugates, prepared by methods that are described in ANTIBODY-AMINODEXTRAN-PHYCOBILIPROTEIN CONJUGATES, parent U.S. patent application Ser. No. 08/847,941 filed May 16, 1997, and incorporated by reference herein. In this parent U.S. patent application, crosslinked conjugates were loaded with substantially more PE than the direct 1:1 antibody-PE conjugates by simultaneously reacting iminothiolane-activated antibody and PE with sulfo-SMCC-activated aminodextran in appropriate molar ratios. Thus, use of these loaded conjugates in saturation amounts in analysis of cell subsets in biological samples resulted in amplification of the PE fluorescence signals from individually-marked cells by a factor of two- to twenty-fold over the PE fluorescence intensity from cells marked with direct antibody-PE conjugates. The amplification of fluorescence was initially demonstrated for marked cells which usually showed dim fluorescence intensity when their receptors were saturated with direct antibody-PE conjugates. Other types of antibody-aminodextran conjugates are described in U.S. Pat. No. 5,527,713 issued Jun. 18, 1996; and U.S. Pat. No. 5,658,741 issued Aug. 19, 1997; see also, European Patent No. 0 594 772 B1.
There remains a need in the art for simple methods to permit greater numbers of cell populations to be distinguished by fluorescent markers.